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中华实验和临床感染病杂志(电子版)

中华实验和临床感染病杂志(电子版) ›› 2020, Vol. 14 ›› Issue (02) : 110 -116. doi: 10.3877/cma.j.issn.1674-1358.2020.02.005

所属专题： 经典病例； 经典病例； 文献；

论著

五例恙虫病东方体感染基因型及其基因变异
姚立农1, 陆群英2,†(), 阮卫1, 占喆2, 朱水荣2, 姜理平2   
  1. 1. 310051　杭州市，浙江省疾病预防控制中心传染病防治所
    2. 310051　杭州市，浙江省疾病预防控制中心微生物所
  • 收稿日期:2019-06-11 出版日期:2020-04-15
  • 通信作者: 陆群英
  • 基金资助:
    2018年浙江省卫生科技项目(No. 2018KY340)

Gene genotypes and variation of 5 cases with Orientia tsutsugamushi infection

Linong Yao1, Qunying Lu2,†(), Wei Ruan1, Zhe Zhan2, Shuirong Zhu2, Liping Jiang2   

  1. 1. Department of Communicable Diseases Control and Prevention, Zhejiang Province Center for Diseases Control and Prevention, Hangzhou 310051, China
    2. Department of Microbiology, Zhejiang Province Center for Diseases Control and Prevention, Hangzhou 310051, China
  • Received:2019-06-11 Published:2020-04-15
  • Corresponding author: Qunying Lu
  • About author:
    Corresponding author: Lu Qunying, Email:
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姚立农, 陆群英, 阮卫, 占喆, 朱水荣, 姜理平. 五例恙虫病东方体感染基因型及其基因变异[J]. 中华实验和临床感染病杂志(电子版), 2020, 14(02): 110-116.

Linong Yao, Qunying Lu, Wei Ruan, Zhe Zhan, Shuirong Zhu, Liping Jiang. Gene genotypes and variation of 5 cases with Orientia tsutsugamushi infection[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2020, 14(02): 110-116.

目的

旨在了解浙闽地区人群恙虫病东方体感染的基因型，并分析其基因变异。

方法

从浙闽地区5例散发恙虫病发热期患者血中分离病原体并进行细胞培养；提取感染细胞DNA，巢式PCR扩增完整恙虫病东方体56-kD型特异性抗原（TSA）基因和热休克蛋白基因（groESL）并测序；采用MEGA 7.0软件，进行序列比对和系统发育分析。

结果

病原学确认5例恙虫病患者，并分离到恙虫病东方体菌株。序列比对表明，5菌株中有2株的56-kD TSA基因和groESL基因100%一致，另2菌株的此二个基因序列一致性亦为100%，分别将两组菌暂时命名为浙江-1型和浙江-2型。56-kDa TSA基因比对和系统发育分析表明，浙江-1型和浙江-2型分别与台湾-A基因型和Gilliam-C基因型亲缘较近（98.45%和98.50%），但有明显变异；另1株菌Wuj/2014与台湾-A基因型高度相似（99.94%）。56-kDa TSA基因各基因型支系的时间树分析表明，台湾-A基因型、浙江-1型和浙江-2型3支系与祖先的分歧时间相对其他原型株晚，尤其是浙江-2型，说明这些基因型或亚型在恙虫病东方体的进化过程中出现较晚。

结论

本研究病例所感染恙虫病东方体基因型不同，可能为未被认识的新的基因亚型，迫切需要进行全基因组测序确认，并探讨其基因变异与人恙虫病严重程度间的关系。

关键词: 立克次体病, 恙虫病, 恙虫病东方体, 基因型, 基因变异
Objective

To investigate the genotype of Orientia tsutsugamushi (O. tsutsugamushi) infection in Zhejiang and Fujian provinces, southeastern China and to analyze the gene variation.

Methods

Strains of O. tsutsugamushi were isolated from the blood of 5 scrub typhus patients of febrile phase and then cell culture were carried out. DNA from the infected cells were extracted and full-length 56-kD type-specific antigen (TSA) gene and full-length heat shock protein gene (groESL) were amplified. The gene products were sequenced. Multiple sequence alignment and phylogenetic analyses were conducted by Mega 7.0 software.

Results

Five febrile patients with O. tsutsugamushi infection were confirmed etiologically and five O. tsutsugamushi strains were isolated. The sequence alignment of 56-kD TSA gene and groESL gene showed that 2 among the 5 strains had 100% identity; while other two strains also had 100% identity of these two genes. These two groups were temporarily named Zhejiang-1 and Zhejiang-2. The 56-kDa TSA gene alignment and phylogenetic analysis showed that Zhejiang-1 was closely related to Taiwan-A genotype (98.45%) and Zhejiang-2 was closely related to Gilliam-C genotype (98.50%), but there were obvious variations. The other 1 strain of Wuj/2014 was significantly similar to Taiwan-A genotype (99.94%). Based on 56-kDa TSA gene, the timetree based on the RelTime method showed that Taiwan-A genotype, Zhejiang-1 and especially Zhejiang-2 genotype were later than other prototypes on the divergence time from their ancestors of each genotypic clade. These genotypes or subtypes emerged later in evolutionary process of O. tsutsugamushi.

Conclusions

There may be unrecognized novel O. tsutsugamushi variants among the patients in this study. The relationship between genetic variation and severity of scrub typhus also should be discussed.

Key words: Rickettsiosis, Scrub typhus, Orientia tsutsugamushi, Genotype, Gene variation
表1 56-kDaTSA基因和groESL基因巢式PCR引物
基因 引物 序列（5′→3′） 来源
groESL gene Gro1 AAGAAGGHGTGATMAC 文献[9]
Gro2 ACTTCMGTAGCACC
SF1 GATAGAAGAAAAGCAATGATG
SR2 CAGCTATTTGAGATTTAATTTG
TF1 ATATATCACAGTACTTTGCAAC
TR2 GTTCCTAACTTAGATGTATCAT
GroF1 GGAAGAAAGTTATATGAAAAGGTGG 本研究
GroF591 TGTACATGGCGATCAATGTCG
GroF1384 TTAAAGTAGTAGCTGTGAAGGCACC
GroR698 GCTGTTCTATTGCTACACATCTACC
GroR2424 GCAAAATACATAACTAGCAACCTTATAC
56-kDa TSA gene F1 GGTTTCGAACGTGTCTTTAAG 本研究
F14 GCGGAAGCTATATATAAGAGCAGT
F754 TGCGTATAAGTATAGCKGATCGTGA
F1425 AGTAGCAGCWGCAGCTGTTAGG
F1490 GATCTTGTTAAATTGMAGCGTCA
R918 CCTTACCATATAGTCAAYACCAGCA
R1527 TTCTTAAWTCCTGCATGACGCT
R1989 GGACCTAGAAGTTATAGCGTACA
R2036 CTGCAAAGGACTTTTAGCTGCT
R2039 AACCCTGCAAAGGACTTTTAGCT
图1 浙江-1型和浙江-2型56 kDa TSA蛋白VDⅠ和VDⅡ氨基酸变异
图2 基于56-kDaTSA基因构建的系统发育树
表2 本研究分离菌株与参考菌株56-kDa TSA基因和groESL基因序列的一致性
菌株 基因 序列号 参考菌株 来源 一致性（%）
Wuj/2014 56-kDa TSA GU120152 KM07 中国台湾省 99.94
EF583448 Taitung-6 中国台湾省 99.81
GU446591 KM11-2 中国台湾省 99.07
KJ001163 Zhou/2012 中国福建省 97.77
M33004 Karp 新几内亚 92.50
AP008981 Ikeda 日本 78.46
groESL KC485340 Zhou/2012 中国福建省 98.69
AP008981 Ikeda 日本 98.23
Jin/2012、Zhou/2012（浙江-1型） 56-kDa TSA KP334159 Panzhihua45 中国四川省 100.00 a
AY283180 China/GDst103 中国广东省 98.58
GU446591 KM11-2 中国台湾省 98.45
EF583448 Taitung-6 中国台湾省 97.96
LS398552 UT76 泰国 97.43
HQ718454 07QN_VN 越南 96·96
M33004 Karp 新几内亚 92.08
AP008981 Ikeda 日本 79.24
groESL AP008981 Ikeda 日本 99.42
M31887 Karp 新几内亚 98.60
Liu/2011、Mu/2013（浙江-2型） 56-kDa TSA EF213093 FPW2049 泰国 98.50
GQ332759 KHC0706a 中国台湾省 98.12
HQ718444 T1116116_KH 柬埔寨 97.84
HQ718460 61QN_VN 越南 97.65
AP008981 Ikeda 日本 95.17
AF050669 Sxh951 中国山西省 94·43
groESL KC688331 O3 日本 99.38
JX188393 Kato 日本 98.59
AP008981 Ikeda 日本 97.81
图3 基于GroESL基因构建的系统发育树（ML）
图4 基于RelTime时间树ML法估计的分歧时间
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